Abstract
One cDNA clone (Cs18h09) encoding Clonorchis sinensis calmodulin (CsCaM) was isolated from our adult cDNA plasmid library. The open reading frame of CsCaM contains 450 bp which encodes 149 amino acids. CsCaM protein comprises four calcium-binding EF-hand motifs. The amino acid sequence of CsCaM shares very high homology with other species. Quantitative RT-polymerase chain reaction (PCR) revealed that CsCaM mRNA was constitutively transcribed in development cycle stages of the parasite, including adult worm, metacercaria, excysted metacercaria, and egg. In addition, recombinant CsCaM (rCsCaM) was expressed as a soluble protein and anti-rCsCaM rat serum could detect CsCaM in the C. sinensis somatic extracts but not in the C. sinensis excretory–secretory products (ESPs). Moreover, immunolocalization assay showed that CsCaM was located in tegument, intestine, pharynx, and eggs. Furthermore, rCsCaM was found to bind calcium ion (Ca2+) and magnesium (Mg2+) in electrophoretic mobility shift assay. Ca2+ binding increased the ability of rCsCaM to bind the hydrophobic fluorescent probe 8-anilinonaphthalene-1-sulphonate, causing a blue shift in the fluorescence emission from 540 to 515 nm with an excitation wavelength of 380 nm and substantial increase in fluorescence intensity but not Mg2+. Collectively, here we showed the basic characterization of CsCaM and inferred that CsCaM could be a Ca2+ sensor protein, and CsCaM may possibly participate in growth and development of adult worm and egg of C. sinensis through binding Ca2+.
Similar content being viewed by others
References
Alirahmi H, Farahnak A, Golmohamadi T, Esharghian MR (2010) Comparative assay of glutathione S-transferase (GSTs) activity of excretory–secretory materials and somatic extract of Fasciola spp parasites. Acta Med Iran 48(6):367–370
Benaim G, Villalobo A (2002) Phosphorylation of calmodulin. Funct Implications 269:3619–3631
Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1(1):11–21
Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, Benbrahim-Tallaa L, Guha N, Freeman C, Galichet L, Cogliano V, WHO International Agency for Research on Cancer Monograph Working Group (2009) A review of human carcinogens—part B: biological agents. Lancet Oncol 10:321–322
Carafoli E, Santella L, Branca D, Brini M (2001) Generation, control, and processing of cellular calcium signals. Crit Rev Biochem Mol Biol 36:107–260
Chin D, Means AR (2000) Calmodulin: a prototypical calcium sensor. Trends Cell Biol 10:322–328
Colhoun LM, Fairweather I, Brennan GP (1998) Observations on the mechanism of eggshell formation in the liver fluke, Fasciola hepatica. Parasitology 116(Pt 6):555–567
Deng C, Sun J, Li X, Wang L, Hu X, Wang X, Chen W, Lv X, Liang C, Li W, Huang Y, Li R, Wu Z, Yu X, Xu J (2012) Molecular identification and characterization of leucine aminopeptidase 2, an excretory–secretory product of Clonorchis sinensis. Mol Biol Rep 39(10):9817–9826
Fraga H, Faria TQ, Pinto F, Almeida A, Brito RM, Damas AM (2010) FH8-a small EF-hand protein from Fasciola hepatica. FEBS J 277(24):5072–5085
Friedberg F (1990) Species comparison of calmodulin sequences. Protein Seq Data Anal 3:335–337
Friedberg F, Rhoads AR (2001) Evolutionary aspects of calmodulin. IUBMB Life 51(4):215–221
Gao Y, Gillen CM, Wheatly MG (2009) Cloning and characterization of a calmodulin gene (CaM) in crayfish Procambarus clarkii and expression during molting. Comp Biochem Physiol B Biochem Mol Biol 152(3):216–225
Gifford JL, Walsh MP, Vogel HJ (2007) Structures and metal-ion-binding properties of the Ca2+ binding helix–loop–helix EF-hand motifs. Biochem J 405:199–221
Grab DJ, Berzins K, Cohen RS, Siekevitz P (1979) Presence of calmodulin in postsynaptic densities isolated from canine cerebral cortex. J Biol Chem 254:8690–8696
Grabarek Z (2006) Structural basis for diversity of the EF-hand calcium-binding proteins. J Mol Biol 359:509–525
Haeseleer F, Imanishi Y, Sokal I, Filipek S, Palczewski K (2002) Calcium-binding proteins: intracellular sensor from the calmodulin superfamily. Biochem Biophys Res Commun 290(2):615–623
Hoeflich KP, Ikura M (2002) Calmodulin in action: diversity in target recognition and activation mechanisms. Cell 108:739–742
Hu S, Law P, Lv Z, Wu Z, Fung MC (2008) Molecular characterization of a calcium-binding protein SjCa8 from Schistosoma japonicum. Parasitol Res 103(5):1047–1053
Huang L, Hu Y, Huang Y, Fang H, Li R, Hu D, Li W, Li X, Liang C, Yu X (2011) Gene/protein expression level, immunolocalization and binding characteristics of fatty acid binding protein from Clonorchis sinensis (CsFABP). Mol Cell Biochem 363(1–2):367–376
Huss HH, Ababouch L, Gram L (2004) Assessment and Management of seafood safety and quality. In: Huss HH (ed) FAO Fisheries Technical Paper, No. 444. Room. pp 60–70
Ikura M (1996) Calcium binding and conformational response in EF hand proteins. Trends Biochem Sci 21:14–17
Ikura M, Osawa M, Ames JB (2002) The role of calcium-binding proteins in the control of transcription: structure to function. Bioessays 24:625–636
James P, Vorherr T, Carafoli E (1995) Calmodulin-binding domains: just two-faced or multifaceted. Trends Biochem Sci 20:38–42
Katsumata T, Kohno S, Yamaguchi K, Hara K, Aoki Y (1989) Hatching of Schistosoma mansoni eggs is a Ca2+/calmodulin-dependent process. Parasitol Res 76(1):90–91
Liang C, Hu XC, Lv ZY, Wu ZD, Yu XB, Xu J, Zheng HQ (2009) Experimental establishment of life cycle of Clonorchis sinensis. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 27:148–150
Lun ZR, Gasser RB, Lai DH, Li AX, Zhu XQ, Yu XB, Fang YY (2005) Clonorchiasis: a key foodborne zoonosis in China. Lancet Infect Dis 5(1):31–41
Lv ZY, Yang LL, Hu SM, Sun X, He HJ, He SJ, Li ZY, Zhou YP, Fung MC, Yu XB, Zheng HQ, Cao AL, Wu ZD (2009) Expression profile, localization of an 8-kDa calcium-binding protein from Schistosoma japonicum (SjCa8), and vaccine potential of recombinant SjCa8 (rSjCa8) against infections in mice. Parasitol Res 104(4):733–743
Lv X, Chen W, Wang X, Li X, Sun J, Deng C, Men J, Tian Y, Zhou C, Lei H, Liang C, Yu X (2012) Molecular characterization and expression of a cysteine protease from Clonorchis sinensis and its application for serodiagnosis of clonorchiasis. Parasitol Res 110(6):2211–2219
Mohamed MM, Shalaby KA, LoVerde PT, Karim AM (1998) Characterization of Sm20.8, a member of a family of schistosome tegumental antigens. Mol Biochem Parasitol 96(1–2):15–25
Niki I, Yokokura H, Sudo T, Kato M, Hidaka H (1996) Ca2+ signaling and intracellular binding proteins. Jo Biochem (Tokyo) 120:685–698
Ohki S, Ikura M, Zhang M (1997) Identification of Mg2+-binding sites and role of Mg2+ on target recognition by calmodulin. Biochemistry 36:4309–4316
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45
Rao KV, Chen L, Gnanasekar M, Ramaswamy K (2002) Cloning and characterization of a calcium-binding, histamine-releasing protein from Schistosoma mansoni. J Biol Chem 277(34):31207–31213
Rim HJ (2005) Clonorchiasis: an update. J Helminthol 79:269–281
Russell SL, McFerran NV, Hoey EM, Trudgett A, Timson DJ (2007) Characterisation of two calmodulin-like proteins from the liver fluke, Fasciola hepatica. Biol Chem 388:593–599
Saimi Y, Kung C (2002) Calmodulin as an ion channel subunit. Annu Rev Physiol 64:289–311
Senawong G, Laha T, Loukas A, Brindley PJ, Sripa B (2012) Cloning, expression, and characterization of a novel Opisthorchis viverrini calcium-binding EF-hand protein. Parasitol Int 61(1):94–100
Shen X, Valencia CA, Gao W, Cotten SW, Dong B, Huang BC, Liu R (2008) Ca(2+)/Calmodulin-binding proteins from the C. elegans proteome. Cell Calcium 43(5):444–456
Sun H, Yin D, Squier TC (1999) Calcium-dependent structural coupling between opposing globular domains of calmodulin involves the central helix. Biochemistry 38(38):12266–12279
Vetter SW, Leclerc E (2003) Novel aspects of calmodulin target recognition and activation. Eur J Biochem 270:404–414
Vichasri-Grams S, Subpipattana P, Sobhon P, Viyanant V, Grams R (2006) An analysis of the calcium-binding protein 1 of Fasciola gigantica with a comparison to its homologs in the phylum Platyhelminthes. Mol Biochem Parasitol 146(1):10–23
Wang CL, Aquaron RR, Leavis PC, Gergely J (1982) Metal-binding properties of calmodulin. Eur J Biochem 124(1):7–12
Wang X, Chen W, Hu F, Deng C, Zhou C, Lv X, Fan Y, Men J, Huang Y, Sun J, Hu D, Chen J, Yang Y, Liang C, Zheng H, Hu X, Xu J, Wu Z, Yu X (2011a) Clonorchis sinensis enolase: identification and biochemical characterization of a glycolytic enzyme from excretory/secretory products. Mol Biochem Parasitol 177:135–142
Wang X, Chen W, Huang Y, Sun J, Men J, Liu H, Luo F, Guo L, LLv X, Deng C, Zhou C, Fan Y, Li X, Huang L, Hu Y, Liang C, Hu X, Xu J, Yu X (2011b) The draft genome of the carcinogenic human liver fluke Clonorchis sinensis. Genome Biol 12(10):R107
Wells KE, Cordingley JS (1991) Schistosoma mansoni: eggshell formation is regulated by pH and calcium. Exp Parasitol 73(3):295–310
Yáñez M, Gil-Longo J, Campos-Toimil M (2012) Calcium binding proteins. Adv Exp Med Biol 740:461–482
Yoshimura T (1993) Monitoring protein conformational changes during membrane fusion. Methods Enzymol 221:72–82
Zhou Z, Xia H, Hu X, Huang Y, Li Y, Li L, Ma C, Chen X, Hu F, Xu J, Lu F, Wu Z, Yu X (2008) Oral administration of a Bacillus subtilis spore-based vaccine expressing Clonorchis sinensis tegumental protein 22.3 kDa confers protection against Clonorchis sinensis. Vaccine 26:1817–1825
Acknowledgments
This work was supported by Development Program of China (973 programs; No. 2010CB530000), the National Natural Science Foundation of China (No. 81101270 and No. 81171602.), and the National Important Sci-Tech Special Projects (2012ZX10004220).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Zhou, J., Sun, J., Huang, Y. et al. Molecular identification, immunolocalization, and characterization of Clonorchis sinensis calmodulin. Parasitol Res 112, 1709–1717 (2013). https://doi.org/10.1007/s00436-013-3329-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-013-3329-z